Supplementary Materials

Antiproliferative activity of berberine in HepG2 cells via inducing

apoptosis and arresting cell cycle

Lele Yang,a Jiliang Cao,a Jinchao Wei,a Jiagang Deng,b Xiaotao Hou,b Erwei Hao,b

Zhengcai Du,b Liang Zou *c and Peng Li *a

a State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese

Medical Sciences, University of Macau, Macau 999078, China

b Collaborative Innovation Center of Research on Functional Ingredients from

Agricultural Residues, Guangxi Key Laboratory of Efficacy Study on Chinese

Materia Medica, Guangxi University of Chinese Medicine, Nanning 530200, China

c Key Laboratory of Coarse Cereal Processing (Ministry of Agriculture and Rural

Affairs), School of Food and Biological Engineering, Chengdu University, Chengdu

610106, China

*Corresponding author:

Email: zouliangcdu@126.com (L. Zhou); pli1978@hotmail.com (P. Li)

Electronic Supplementary Material (ESI) for Food & Function.This journal is © The Royal Society of Chemistry 2021

Fig. S1. (A) Principal component analysis plot illustrating the sample distribution of

blank control (B, red) and berberine-treated (BER, green) group. (B) Heatmap of all

downregulated proteins. (C) Top 10 significant enriched KEGG pathways and (D) top

8 enriched Reactome pathways. (E) Protein-protein interaction network of altered

proteins assessed with the STRING database. Proteins labeled in red and blue color

were enriched in the mitochondria and metabolism pathways.

Fig. S2. (A) Overrepresented pathways from REACTOME pathway analysis of the

altered proteins. Expression of mitochondrial dysfunction-mediated protein, including

(B) ATP5O, (C) ATP5D, (D) NDUFAB1, (E) COX5B. ATP5O, ATP synthase

subunit O; ATP5D, ATP synthase subunit delta; NDUFAB1, NADH dehydrogenase

(ubiquinone) 1 beta subcomplex; COX5B, Cytochrome c oxidase subunit 5B.

Fig. S3. (A, B) Box plots of AKAP12 expression (p ≤ 0.001) and AFP expression (p ≤

0.001) in normal and tumor samples from GSE112790 dataset. (C) Correlation

analysis between expression levels of AKAP12 and AFP (p = 0.005, r=-0.2). (D)

Spearman correlation analysis of the expression of AKAP12 and its methylation

levels using LinkedOmics. (E) Dot plot of the enriched GO terms using the

upregulated genes obtained from TCGA LIHC dataset.

Fig. S4. The protein-sequence coverage of AKAP12 observed using Proteome

Discoverer software (version 2.3.0).

Images in support of the representative western blots data shown in Figure 6. (A, B)

Western blot analysis of AKAP12 and Cyclin D1 expression. (C) Co-

immunoprecipitation of AKAP12 and Cyclin D1 was visualized using immunoblots.

Table S1. The hub target proteins of berberine (ranked according to Clustering

Coefficient).

name Glay Cluster

Average ShortestPath Length

BetweennessCentrality Closeness Centrality Clustering

Coefficient Degree

SHC1 1 2.506 0.006 0.399 0.189 61

SMAD4 3 2.507 0.005 0.399 0.170 48

PTK2 1 2.521 0.006 0.397 0.156 47

RAF1 1 2.452 0.005 0.408 0.146 47

HDAC1 3 2.480 0.008 0.403 0.120 59

LCK 1 2.485 0.005 0.402 0.120 54

BRCA1 3 2.362 0.009 0.423 0.119 68

BCL2 5 2.523 0.006 0.396 0.119 39

PIK3R1 1 2.426 0.013 0.412 0.117 72

SMAD2 3 2.360 0.010 0.424 0.116 62

STAT3 3 2.382 0.010 0.420 0.115 64

JUN 3 2.452 0.008 0.408 0.114 73

XRCC6 3 2.520 0.007 0.397 0.114 33

KAT5 3 2.474 0.005 0.404 0.113 36

RELA 3 2.492 0.007 0.401 0.112 68

RXRA 3 2.496 0.007 0.401 0.112 56

SMAD3 3 2.288 0.016 0.437 0.109 79

GRB2 1 2.471 0.011 0.405 0.107 83

CAV1 1 2.458 0.012 0.407 0.105 45

PRKCD 1 2.462 0.007 0.406 0.102 58

CCND1 3 2.527 0.011 0.396 0.101 40

Table S2. Apoptosis and cell cycle-related enrichment pathways.

term ID term description observed gene count

background gene count

false discovery rate matching proteins in your network (labels)

GO:0042981 regulation of apoptotic process 32 1501 6.63E-23ABL1,AKT1,AR,BCL2,BRCA1,CASP3,CAV1,CDK1,CDK5,CREBBP,CSNK2A1,CTNNB1,EGFR,ESR1,FYN,GSK3B,HDAC1,JUN,LCK,PIK3R1,PRKCA,PRKCD,PTK2,RAF1,RELA,SHC1,SMAD3,SRC,STAT3,TP53,YWHAG,YWHAZ

GO:0043066 negative regulation of apoptotic process 27 859 1.19E-22 ABL1,AKT1,AR,BCL2,BRCA1,CASP3,CAV1,CDK1,CSNK2A1,CTNNB1,EGFR,FYN,GSK3B,HDAC1,JUN,PIK3R1,PRKCA,PRKCD,PTK2,RAF1,RELA,SHC1,SMAD3,SRC,STAT3,TP53,YWHAZ

GO:2001233 regulation of apoptotic signaling pathway 18 388 2.53E-17 AKT1,AR,BCL2,BRCA1,CAV1,CSNK2A1,CTNNB1,FYN,GSK3B,LCK,PRKCD,RAF1,RELA,SMAD3,SRC,TP53,YWHAG,YWHAZ

GO:0006915 apoptotic process 22 915 7.26E-16 ABL1,AKT1,BCL2,BRCA1,CASP3,CAV1,CDK1,CDK5,CSNK2A1,EP300,GSK3B,JUN,MAPK1,MAPK3,NR3C1,PIK3R1,PRKCA,PRKCD,RAF1,RB1,SMAD3,TP53

GO:0097190 apoptotic signaling pathway 14 295 1.49E-13 ABL1,BCL2,BRCA1,CASP3,CAV1,EP300,GSK3B,JUN,PIK3R1,PRKCA,PRKCD,RAF1,SMAD3,TP53

GO:0043065 positive regulation of apoptotic process 16 604 6.72E-12 ABL1,AKT1,BCL2,CASP3,CAV1,CDK5,CTNNB1,GSK3B,JUN,LCK,PRKCD,SMAD3,SRC,TP53,YWHAG,YWHAZ

GO:2001234 negative regulation of apoptotic signaling pathway 10 218 1.29E-09 AKT1,AR,BCL2,BRCA1,CSNK2A1,CTNNB1,FYN,RAF1,RELA,SRC

GO:2001236 regulation of extrinsic apoptotic signaling pathway 9 158 1.87E-09 AKT1,AR,BCL2,BRCA1,CAV1,FYN,RAF1,RELA,SRC

GO:2001237 negative regulation of extrinsic apoptotic signaling pathway 8 104 2.17E-09 AKT1,AR,BCL2,BRCA1,FYN,RAF1,RELA,SRC

GO:0045930 negative regulation of mitotic cell cycle 11 243 1.79E-10 ABL1,BCL2,BRCA1,CCND1,CDK1,CTNNB1,EGFR,EP300,RB1,SMAD3,TP53

GO:1901990 regulation of mitotic cell cycle phase transition 12 351 4.23E-10 AKT1,BCL2,BRCA1,CCND1,CDK1,EGFR,EP300,HSP90AA1,PRKACA,RB1,TP53,YWHAG

GO:0010564 regulation of cell cycle process 15 684 4.55E-10 AKT1,BCL2,BRCA1,CCND1,CDK1,CDK5,CSNK2A1,CTNNB1,EGFR,EP300,HSP90AA1,PRKACA,RB1,TP53,YWHAG

GO:0045787 positive regulation of cell cycle 12 376 8.59E-10 ABL1,AKT1,BRCA1,CCND1,CDK1,EGFR,EP300,PRKACA,PRKCA,RB1,SRC,TP53

GO:0045786 negative regulation of cell cycle 13 517 2.04E-09 ABL1,BCL2,BRCA1,CASP3,CCND1,CDK1,CDK5,CTNNB1,EGFR,EP300,RB1,SMAD3,TP53

GO:2000045 regulation of G1/S transition of mitotic cell cycle 8 148 2.68E-08 AKT1,BCL2,CCND1,CDK1,EGFR,EP300,RB1,TP53

GO:0071156 regulation of cell cycle arrest 7 111 9.42E-08 BRCA1,CCND1,CDK1,CDK5,EP300,PRKACA,TP53

GO:0022402 cell cycle process 13 890 8.16E-07 ABL1,BRCA1,CCND1,CDK1,CTNNB1,EP300,HSP90AA1,PRKACA,PRKCA,RB1,SMAD3,TP53,YWHAG

GO:1901991 negative regulation of mitotic cell cycle phase transition 7 160 8.70E-07 BCL2,BRCA1,CCND1,CDK1,EP300,RB1,TP53

GO:2000134 negative regulation of G1/S transition of mitotic cell cycle 6 97 1.03E-06 BCL2,CCND1,CDK1,EP300,RB1,TP53

GO:0000278 mitotic cell cycle 11 628 1.49E-06 ABL1,BRCA1,CCND1,CDK1,EP300,HSP90AA1,PRKACA,PRKCA,RB1,TP53,YWHAG

hsa04210 Apoptosis 10 135 4.83E-12 AKT1,BCL2,CASP3,JUN,MAPK1,MAPK3,PIK3R1,RAF1,RELA,TP53

hsa04215 Apoptosis - multiple species 2 31 0.0037 BCL2,CASP3

hsa05200 Pathways in cancer 31 515 4.81E-35ABL1,AKT1,AR,BCL2,CASP3,CCND1,CREBBP,CTNNB1,EGFR,EP300,ESR1,GRB2,GSK3B,HDAC1,HSP90AA1,JUN,MAPK1,MAPK3,PIK3R1,PRKACA,PRKCA,PTK2,RAF1,RB1,RELA,RXRA,SMAD2,SMAD3,SMAD4,STAT3,TP53

hsa05225 Hepatocellular carcinoma 17 163 3.07E-22 AKT1,CCND1,CTNNB1,EGFR,GRB2,GSK3B,MAPK1,MAPK3,PIK3R1,PRKCA,RAF1,RB1,SHC1,SMAD2,SMAD3,SMAD4,TP53

hsa04110 Cell cycle 14 123 5.97E-19 ABL1,CCND1,CDK1,CREBBP,EP300,GSK3B,HDAC1,RB1,SMAD2,SMAD3,SMAD4,TP53,YWHAG,YWHAZ

hsa04217 Necroptosis 3 155 0.0081 BCL2,HSP90AA1,STAT3

Table S3. Dysregulated proteins in berberine-treated group compared to control (ranked according to fold change).

No. Unique peptides Sequence coverage [%] MS/MS count Log Student's T-test p-value Student's T-test Difference Gene names1 16 45.2 114 4.9 -4.7 AFP2 9 32.6 30 5.8 -2.8 FGG3 8 70.1 55 4.4 -2.6 SLIRP4 6 6.5 28 3.9 -2.5 FN15 8 8.5 26 4.2 -2.3 A2M6 24 44 199 6.6 -2.3 TF7 3 31.8 37 3.6 -2.1 LYZ8 8 26.7 52 2.9 -1.9 PEG109 6 25.7 60 3.5 -1.9 ACADM10 4 15.2 96 3.8 -1.8 SLC39A1411 6 15.1 16 3.3 -1.8 P4HA112 3 14.5 16 2.4 -1.8 HSDL213 4 24.7 25 1.8 -1.7 MT-CO214 2 15 25 3.1 -1.7 METTL7A15 2 21 12 4.2 -1.7 GLRX516 5 23.7 34 1.6 -1.7 NDUFAB117 12 13.9 19 3.6 -1.7 SEC31A18 4 10.1 90 1.8 -1.7 SLC16A319 3 12.9 9 2.5 -1.5 SDHB20 4 8.2 10 1.4 -1.5 RPS6KA321 2 19.1 18 2.0 -1.5 PPT122 4 57 12 2.2 -1.5 COX6B123 3 53.1 47 2.7 -1.5 CYB5A24 3 13.9 62 2.3 -1.5 AHSA125 2 9.3 17 2.3 -1.4 COX5B

26 7 7.3 19 2.3 -1.4 UGGT127 2 11.9 22 1.6 -1.4 ACOT128 5 27.9 23 1.4 -1.3 C14orf16629 3 5.8 8 2.3 -1.3 GLDC30 6 18.8 41 1.9 -1.3 IGF2BP331 2 7.5 3 1.5 -1.3 DLAT32 11 31.6 57 4.2 -1.3 HADHB33 6 19.8 13 2.5 -1.3 PECI34 2 47.6 74 3.1 -1.2 HIST3H2BB35 2 17.2 7 1.8 -1.2 RAC136 2 11.6 19 2.7 -1.2 FKBP337 5 15.1 18 3.3 -1.2 PGM138 18 42.5 173 3.9 -1.2 PCK239 3 37.5 79 1.8 -1.2 ATP5D40 8 54.5 71 4.0 -1.2 ATP5O41 6 14.7 9 1.4 -1.2 RANGAP142 2 61.9 30 1.4 -1.2 RPL3843 20 52.4 480 2.9 -1.1 TUFM44 4 48.6 110 1.9 -1.1 TOMM2245 16 20.4 30 1.7 -1.1 COPA46 4 9.7 7 2.7 -1.1 TM9SF447 8 33.7 283 2.9 -1.1 CTSD48 3 12.4 25 2.1 -1.1 PAK249 10 43 135 3.0 -1.1 UQCRC250 4 40.7 150 3.0 -1.0 COX5A51 6 48.7 168 1.7 -1.0 HSD17B1052 10 44.2 91 2.1 -1.0 PYCR153 0 55.2 152 1.3 -1.0 H2AFX54 7 32.9 61 4.6 -1.0 RALY

55 8 33.5 52 4.1 -1.0 ACAA256 4 25.6 43 1.5 -1.0 SEC22B57 3 57.4 48 3.7 -1.0 ATP5J2-PTCD158 3 44.8 18 2.3 -1.0 NDUFA559 0 66.2 1095 4.4 -1.0 HIST1H2AJ60 3 44.4 56 3.4 -0.9 ATP5J61 5 37.3 118 3.9 -0.9 RBBP462 4 4.9 16 1.6 -0.9 SUPT16H63 3 61.9 1271 4.2 -0.9 GLUD164 2 41.6 55 1.3 -0.9 CFL265 11 29.6 69 2.4 -0.9 CAT66 3 18.4 6 1.8 -0.9 PGRMC267 17 35.7 103 4.1 -0.9 ANXA668 14 56.7 735 4.0 -0.9 HEL-S-3969 14 50.9 167 3.2 -0.9 ALDH270 7 45.4 198 3.2 -0.9 BSG71 2 16.6 28 1.3 -0.9 RPL2972 10 52.1 271 3.1 -0.9 ETFA73 2 6.2 3 3.1 -0.9 LBR74 3 30.9 276 2.3 -0.9 PPIF75 8 37.9 257 1.9 -0.9 DHRS276 7 33.8 145 3.4 -0.9 DNAJA177 8 32.4 140 3.4 -0.9 AGMAT78 2 47.6 1165 4.7 -0.8 HIST1H2BM79 1 9.7 140 1.7 -0.8 NOLC180 16 47.3 170 2.6 -0.8 FDXR81 6 44.4 240 2.2 -0.8 RPL1182 3 13.7 11 1.6 -0.8 DAP383 5 20 94 1.3 -0.8 U2AF1

84 4 35.4 10 4.9 -0.8 BAX85 13 32.7 163 2.8 -0.8 FACL486 4 21.7 16 1.8 -0.8 ZMPSTE2487 24 45.6 362 3.5 -0.8 HADHA88 8 31.3 133 3.5 -0.7 CS89 9 41.8 426 2.9 -0.7 ERP2990 4 19.4 32 2.7 -0.7 BUB391 13 33.3 109 2.1 -0.7 HSD17B492 3 13.6 10 1.5 -0.7 GALK193 36 75.6 3914 6.8 -0.7 P4HB94 3 49.4 29 1.4 -0.7 DYNLL195 33 56.3 853 3.6 -0.7 PDIA496 33 28.5 511 4.0 -0.7 RRBP197 7 22.1 54 5.0 -0.7 FDFT198 3 51 8 2.1 -0.7 SEC61B99 13 44.2 199 2.2 -0.7 TXNDC5100 4 5.9 29 3.1 -0.7 SCP2101 9 33.1 296 1.5 -0.6 ACADSB102 8 34.5 86 2.5 -0.6 ETFB103 10 55.2 494 2.4 -0.6 PCNA104 6 23.2 24 3.8 -0.6 PDHB105 4 11.7 131 1.5 -0.6 EWSR1106 5 22.1 12 3.0 -0.6 PRMT1107 3 1.6 15 1.4 -0.6 SRRM2108 43 25 503 3.6 0.6 FLNA109 20 56.6 260 3.7 0.6 ACTN1110 4 20.4 109 1.6 0.6 RPL13111 4 4.8 5 1.4 0.6 IQGAP1112 4 31.2 233 2.2 0.6 CLIC4

113 15 48.4 176 2.0 0.6 GSR114 17 5.7 38 1.4 0.6 DYNC1H1115 18 43.9 208 2.9 0.6 YARS116 4 28.5 28 1.5 0.6 VAPA117 24 60.1 464 1.9 0.7 TCP1118 4 26.1 168 2.2 0.7 PSMA2119 14 58.1 159 2.3 0.7 RNH1120 17 83 1150 4.2 0.7 CLIC1121 3 7.6 10 1.4 0.7 ACY1122 10 42.2 176 2.3 0.7 PLIN3123 20 62.9 1003 2.7 0.7 CAP1124 8 13.1 31 2.5 0.7 ITGB1125 14 62.7 764 1.6 0.7 YWHAE126 23 62.5 768 4.4 0.7 PGD127 1 80.5 2996 2.7 0.7 AKR1C1128 4 94.7 1507 1.4 0.7 RPLP1129 8 51.4 144 1.7 0.8 CAPZA1130 53 35.5 728 5.4 0.8 FLNB131 4 11.7 4 2.0 0.8 SEPT7132 2 10.6 12 2.8 0.9 NDRG3133 4 33 23 1.5 0.9 UBE2K134 22 42.7 270 6.1 0.9 GFPT1135 101 58.3 1779 7.7 0.9 SPTBN1136 6 49.5 325 1.9 0.9 TPD52L2137 9 17.1 482 4.7 0.9 SLC2A1138 23 23.9 252 2.2 0.9 MYH9139 5 18.1 28 2.5 0.9 CORO1C140 4 6.2 5 1.4 0.9 EIF5B141 7 48.8 159 2.9 1.0 TPM4

142 3 27.3 5 1.3 1.0 GMFB143 2 11.5 11 2.4 1.2 FBXO2144 3 9.7 12 1.8 1.2 HPD145 3 32.2 12 2.8 1.2 RBM8A146 9 14.9 30 1.8 1.2 VCL147 4 45.2 37 2.1 1.3 PCBD148 13 60.5 761 2.8 1.3 SFN149 4 33.8 81 2.5 1.4 CHCHD2150 2 22.3 16 1.4 1.5 LZIC151 6 58.5 121 4.2 1.5 LGALS1152 5 71.6 624 3.4 1.5 S100P153 4 64.8 47 3.8 1.8 S100A11154 4 20.9 18 4.0 1.8 ARG2155 17 28.6 164 5.9 2.1 ACSL5156 9 40.2 184 6.1 2.6 SQSTM1157 14 41.3 171 2.8 2.6 TXNRD1158 12 45.1 215 6.3 3.5 GDF15159 7 22.6 116 6.1 3.8 CYP1A1160 28 30.7 174 6.5 4.4 AKAP12